T cell receptor (TCR) gene rearrangement through V(D)J recombination provides the structural basis for adaptive immunity. This gene rearrangement event is unique to the lymphoid system and yields a rich repertoire of T cells which are capable of recognizing a diverse array of peptide antigens during immune responses. Very little is known about the regulatory molecules that control the precise timing and selectivity of transcription of individual TCR genes during T cell development in the thymus. Recent works have shown that the transcription factors E2A and HEB play important roles in initiating lymphoid specific V(D)J recombination events. Genetic studies carried out in our own laboratory further suggested that E2A/HEB are directly involved in gene rearrangement at the TCR beta locus. In this proposal, we plan to specifically investigate the role of E2A/HEB in TCR beta gene transcription and rearrangement during T cell development. First, we hypothesize that E2A and HEB transcription factors are directly involved in TCR V beta gene transcription to ensure that all the V genes have a chance to be used in rearrangement. Second, this E2A/HEB activity must be down regulated subsequently at the DP stage to prevent biallelic expression of the TCR beta gene, a process known as allelic exclusion. We have developed several mouse models to allow both genetic and biochemical investigations of E2A/HEB function in T cell development. In particular, an E2A knockin mouse expressing tagged E2A molecules will be used in chromatin immunoprecipitation assays to determine where and when E2A functions in the TCR beta locus. Gene knockout mice will be used to determine the causal link between E2A/HEB and TCR gene expression. While the proposed research focuses on the dissection of TCR beta gene locus, the methods and concepts are generally applicable to understanding other E2A/HEB target genes identified in T cells. Most E2A/HEB targets, including TCR beta, preT alpha, and TCR alpha genes, must be coordinately expressed to ensure the proper progression and completion of T cell development. Thus, we will also conduct a genome-based study to broaden our understanding of the coordinate gene regulation events during T cell development.